Cholate solubilized proteins from dimethylmaleic anhydride extracted rat adipocyte plasma membranes have been shown to retain cytochalasin B-sensitive D-glucose transport when reconstituted in the presence of exogenous phospholipids (J. Biol. Chem. 252:8341, 1977). In the present studies, cholate solubilized proteins were subjected to HA and Sepharose 6B chromatography prior to reconstitution in an attempt to further purify the transport protein(s). HA columns equilibrated in 10mM KH2PO4-K2HPO4, 0.5% Na cholate, pH 7.4 were eluted with a linear phosphate gradient. One peak which contains 20-40% of the protein and elutes at approximately 200 mM phosphate, contains all the transport activity. Using a Sepharose 6B column equilibrated with 100 mM NaCl, 10mM NaH2PO4-Na2HPO4, 0.5% Na cholate, pH 7.4, all the activity is found in the higher molecular weight fractions (60-80A). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these column fractions indicated that transport activity did not correlate with an increase in purification of any of the major protein bands (94,000, 78,000, and 64,000 daltons) of the cholate-solubilized membrane protein. It is concluded that the transport protein(s) 1) cannot be the major component of any one of these bands and 2) behaves as a high molecular weight complex in cholate. Further purification steps are in progress.